Isolation and amino acid sequence of two new PR-4 proteins from wheat

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwin1. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced geneswin1 andwin2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 andwin1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product ofwin1,win2, andhev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.     

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifungal activity toward the wide-host-range pathogenBotrytis cinerea and the wheat-specific pathogenic fungi of wheatFusarium culmorum andFusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.     

Conserved F84 and P86 residues in alphaB-crystallin are essential to effectively prevent the aggregation of substrate proteins

Previously, we have shown that residues 73-92 (sequence DRFSVNLDVKHFSPEELKVK) in alphaB-crystallin are involved in preventing the formation of light scattering aggregates by substrate proteins. In this study, we made single substitutions of three conserved amino acid residues (H83 --> A, F84 --> G, and P86 --> A) and a nonconserved amino acid residue (K90 --> C) in the functional region of alphaB-crystallin and evaluated their role in anti-aggregation activity. Mutation of conserved residues led to changes in intrinsic tryptophan intensity, bis-ANS binding, and in the secondary and tertiary structures. The H83A mutation led to a twofold increase in molar mass, while the other mutants did not produce significant changes in the molar mass when compared to that of wild-type protein. The chaperone-like activity of the H83A mutant was enhanced by 15%-20%, and the chaperone-like activity of F84G and P86A mutants was reduced by 50%-65% when compared to the chaperone-like activity of wild-type alphaB-crystallin. The substitution of the nonconserved residue (K90 --> C) did not induce an appreciable change in the structure and function of the mutant protein. Fluorescence resonance energy transfer (FRET) assay demonstrated that destabilized ADH interacted near the K90 region in alphaB-crystallin. The data show that F84 and P86 residues are essential for alphaB-crystallin to effectively prevent the aggregation of substrate proteins. This study further supports the involvement of the residues in the 73-92 region of alphaB-crystallin in substrate protein binding and chaperone-like action.     

Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11,180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75-76; 91-93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.     

Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.     

The primary sequence of human pancreatic colipase

The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase₈₆) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.     

Structure-function relationships in the stereospecific and manganese-dependent 3,4-dihydroxyphenylalanine/tyrosine-sulfating activity of human monoamine-form phenol sulfotransferase,SULT1A3

The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their d-form enantiomers and can be stimulated dramatically by Mn(2+). This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise >93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn(2+) of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84-86 and 84-90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84-90 of variable Region I, in the stereospecificity in the absence of Mn(2+). Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn(2+). These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.     

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.     

Primary structures of dog and cat amyloid A proteins: comparison to human AA

1. The complete amino acid sequences of canine and feline amyloid A (AA) proteins were determined and compared with the sequence of human AA protein. 2. The dog and cat AA proteins were 84% homologous with human AA through residue 69. 3. Between the residues which correspond to 69 and 70 in the human sequence, the dog and cat proteins had an insertion of eight amino acids after which homology with human AA resumed. 4. While human AA commonly ends at position 76, the carboxyl termini of dog and cat AA proteins corresponded to position 86 in the sequence of the precursor protein-serum amyloid A. 5. These results are particularly interesting with respect to evolution of the serum amyloid A gene family.     

Recombinant Wheat Antifungal PR4 Proteins Expressed in Escherichia coli

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

Primary structure of human urinary prokallikrein

The complete amino acid sequence of human urinary prokallikrein has been determined by amino acid analysis and sequence determination of peptide fragments obtained from chemical and enzymological cleavages of kallikrein and by comparison of the N-terminal sequence of prokallikrein with that of kallikrein, the active form. Prokallikrein was a single chain polypeptide which comprised 238 amino acid residues of kallikrein and 7 amino acid residues of the propeptide. The sequence, Asn-X-Thr(Ser), which is a common glycosylation site was found at positions 78-80, 84-86, and 141-143. Two trypsin-susceptible sites were identified. One is the Arg(-1)-Ile(1) bond and the other is the Arg (87)-Gln(88) bond. The sequence of human urinary kallikrein was identical with that of human pancreatic and kidney kallikreins (Fukushima, D. et al. (1985) Biochemistry 24, 8037-8043; Baker, A.R. & Shine, J. (1985) DNA 4, 445-459), which were predicted from the nucleotide sequences of cDNAs. The primary structure of human urinary kallikrein is homologous to those of the other animal kallikreins and kallikrein-related proteins. Key amino acid residues, His(41), Asp(96), and Ser(190), required for catalytic activity and Asp (184) required for kallikrein-type specificity are completely conserved. The results show that human urinary prokallikrein and kallikrein are of tissue type and they are excreted in urine without any modification.     

Sequences outside a minimal immunodominant site exert negative effects on recognition by staphylococcal nuclease-specific T cell clones

In recent years, synthetic peptides have been utilized extensively to characterize the minimal essential immunodominant sites on model protein Ag. However, little work has focused on the effect that sequences flanking these minimal recognition sites may exert on T cell recognition. Previous work with staphylococcal nuclease (Nase) demonstrated that I-Ek-restricted clones recognize the peptide 81-100, whereas I-Ab-restricted clones recognize the over-lapping but non-cross-reacting peptide 91-110. Further analysis with 15 or 10 residue peptides within the region 81-110 reveals that the minimal sequence capable of stimulating I-Ek-restricted clones is contained within the decapeptide 91-100. Addition of residues 86-90, to give the peptide 86-100, enhanced the recognition substantially, whereas addition of residues 101-105 produced a 91-105 peptide with no stimulatory ability. These results suggest that interactions between the antigenic peptide 91-100 and residues within the flanking 101-105 sequence have negative consequences for presentation of the immunodominant epitope to T cell clones. Introduction of single amino acid substitutions within 91-105 produced peptides that induce responses comparable to those seen with 91-100. These results are consistent with the suggestion of negative interactions between the minimal immunodominant site and flanking sequences in that single residue substitutions may remove these negative interactions and lead to restoration of stimulatory ability. The negative effect of flanking sequences on T cell recognition of immunodominant sites presents new considerations for development of synthetic vaccines as well as for understanding the biology of Ag processing and presentation.     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.     

COMPARATIVE STUDIES OF GUINEA PIG AND BOVINE MYELIN BASIC PROTEINS. PARTIAL CHARACTERIZATION OF CHEMICALLY DERIVED FRAGMENTS AND THEIR ENCEPHALITOGENIC ACTIVITIES IN LEWIS RATS

Guinea pig and bovine myelin basic proteins were chemically cleaved at the carboxyl peptide bonds of methionyl and tryptophanyl residues to yield several fragments. Comparison of the bovine fragment consisting of the first 20 residues of the protein with the corresponding guinea pig fragment showed that the latter differs in containing histidine and glycine (one residue of each), an additional threonyl residue, and one fewer alanyl residues. Comparison of the bovine fragment consisting of the C-terminal 54 residues of the protein (residues 117-170) with the corresponding guinea pig fragment showed that the latter differs in containing one fewer histidyl and leucyl residues and an additional phenylalanyl residue. Tests of encephalitogenic activity in Lewis rats showed that these two fragments from both species were much less active, on a molar basis, than the uncleaved protein. On the other hand, examination of the bovine fragments consisting of residues 1-116 and 21-116 and the corresponding fragments obtained from the guinea pig protein revealed activity at least as high as that of the respective uncleaved proteins.     

Secretory cell actin-binding proteins: identification of a gelsolin- like protein in chromaffin cells

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.     

Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera)

Four closely related cDNA clones encoding laccase isoenzymes from xylem tissues of yellow-poplar (Ltlacc2.1–4) were identified and sequenced. The inferred yellow-poplar laccase gene products were highly related to one another (79–91% at the amino acid level) and showed significant similarity to other blue copper oxidases, especially with respect to the copper-binding domains. The encoded proteins had N-terminal signal sequences and 17–19 potential N-linked glycosylation sites. The mature proteins were predicted to have molecular masses of ca. 61 kDa (unglycosylated) and high isoelectric points (pI 9.3–9.5). The canonical copper ligands were conserved, with the exception of a Leu residue associated with the axial position of the Type-1 cupric ion. The residue at this position has been proposed to influence the redox potential of Type-1 cupric ions. Northern blot analysis revealed that the yellow-poplar laccase genes are differentially expressed in xylem tissues. The genes were verified as encoding active laccases by heterologous expression in tobacco cells and demonstration of laccase activity in extracts from transformed tobacco cell lines.     

Recombinant wheat antifungal PR4 proteins expressed in Escherichia coli.

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.